In recent years, 6-substituted aminopurines have assumed considerable biochemical significance. Some compounds of this type promote plant growth and belong to the group of growth regulators termed cytokinins (Letham, Ann. Rev. Plant. Physiol. 18, 349, 1967). In cytokinin bioassays based on induction of cell division in plant tissue cultures, the most active compounds is the naturally occurring cytokinin trans-zeatin (6-((E)-4-hydroxy-3-methylbut-2-enylamino)purine: Letham, Planta 74:228, 1967). Cytokinins closely related to zeatin occur as bases in soluble RNA (Skoog et al., Science 154:1354, 1966). In the serine and tyrosine RNAs of yeast, plants, and animals the cytokinin is adjacent to the anticodon. The growth of mammalian cell cultures is inhibited by certain N6-substituted adenosines with cytokinin activity (Grace et al., Proc. Am. Assoc. Cancer Res. 8:23, 1967). After the discovery of kinetin (Miller et al., J. Amer. Chem. Soc. 77:1392, 1955), there was a flurry of activity that led to the finding of 6-benzylaminopurine (BA), an active and easily obtainable cytokinin. Much research into cytokinin physiology was subsequently done with this substance.
Alkylation of natural cytokinins at position 9 of the purine nucleus may occur in plants. Lupinic acid, a zeatin conjugated at N9 with the amino acid alanine, was the first detected metabolite of this type (MacLeod et al., J. Org. Chem. 41: 3959, 1976; Duke et al., Phytochemistry 18:819, 1978; Parker et al., Planta 142:239, 1978). Later, the corresponding 9-alanyl derivative was identified as a metabolite of BA in bean seedlings (Letham et al., Phytochemistry 17:2053, 1979; Zhang et al., J. Plant Growth Regul. 8:181, 1989). Like 9-alanyl zeatin, it exhibited low biological activity and higher stability than the corresponding bases (Parker et al., Planta 142:239, 1978; Palni et al., Planta 160:242, 1984; Zhang et al., J. Plant Growth Regul. 8:181, 1989). The minimisation of BA conjugation has been of both biotechnological and agronomic interest for some time (see, e.g., Zhang et al., J. Plant Growth Regul. 8:181, 1989; Werbrouck et al., Physiol. Plant. 98:291, 1996). 9-Substituted BA derivatives which slowly release free BA may possess enhanced cytokinin activities (e.g., senescence retarding, in vitro morphogenesis, cell division stimulating, etc.) since these compounds are not directly subject to inactivation by conjugation.
A number of 9-substituted cytokinin derivatives have been reported but their structure activity relationships still remain an enigma. The most effective 9-alkyl derivatives developed so far are 9-(2-tetrahydropyranyl)-BA (van Overbeek et al., Science 156:1497, 1967) and 9-(2-tetrahydrofuranyl)-BA (Zhang et al., J. Plant Growth Regul. 8:181, 1989), which both proved to be considerably more active than BA in evoking several growth responses. Since the tetrahydropyranyl group is readily cleaved by acid hydrolysis, it had been suggested that the high biological activity of 9-(2-tetrahydropyranyl)-N6-alkyladenines is probably a consequence of slow cleavage of the 9-substituent (Young et al., Phytochemistry 8:1199, 1969). Subsequently, Fox et al. (Plant Physiol. 47:275, 1971) studied the metabolism of the less active 9-methyl-BA in tobacco and soybean callus tissue and demonstrated rapid conversion to several products. The metabolites were not identified definitively, although it was proposed that conversion to free BA occurred. Pietraface et al., (Physiol. Plant. 53:249, 1981) examined the metabolism of 9-methyl-BA in germinating lettuce seed and suggested formation of BAR and BAR5′P on the basis of chromatographic data. Nevertheless, free BA was not detected. Finally, the application of a 9-(2-tetrahydropyranyl)- and a 9-(2-tetrahydrofuranyl)-BA, assessed for their ability to retard soybean leaf senescence, led to release of free BA (Zhang et al., J. Plant Growth Regul. 8:181, 1989). Both compounds were also debenzylated to adenine substituted with 9-tetrahydropyranyl and 9-tetrahydrofuranyl moiety, respectively. The observed high activity of these 6-benzylamino-9-alkylpurines could be a consequence of their ability to release the free base and to maintain an optimal concentration of the free base over a prolonged period (Zhang et al., J. Plant Growth Regul. 8:181, 1989). Thus, the susceptibility to enzymatic dealkylation is probably the critical factor determining the biological activity of 9-alkyl cytokinins. Hence the less active compounds (Kende et al., Plant Physiol. 43: 1244, 1968; Young et al., Phytochemistry 8:1199, 1969; Corse et al., J. Plant Growth Reg. 8:211, 1989; Motyka et al., SPB Acad Publ., ISBN 90-5103-066-5, p. 215, 1992) are probably not susceptible to cleavage of the 9-substituent and exhibit low or zero activity because of their stability. The enhanced activity of 9-alkyl-BAs relative to those of BA, can be consequently attributed to their ability to gradually release the active free base.
This invention provides growth-regulatory, differentiating, and antisenescent cytokinin analogues having improved selectivity and efficiency index (i.e., that are less toxic yet more efficacious) than analogues known heretofore.